Using contact-dependent three-dimensional coculture systems and serum-free conditions, we compared the ability of estrogen receptor (ER)-alpha(+) tamoxifen-sensitive premalignant (EIII8) or tumorigenic (MCF-7), ER-alpha(+) tamoxifen-resistant (EIII8-TAM(R)) or ER-alpha(-) MDA-MB-231 breast cancer cells to interact and undergo epithelial morphogenesis on association with breast tumor-derived fibroblasts. Although all breast cancer cell lines interacted with tumor fibroblasts, EIII8 and its intrinsically tamoxifen-resistant counterpart EIII8-TAM(R) cells were most receptive and responded with dramatic, albeit, aberrant epithelial morphogenesis. EIII8 cells underwent epithelial morphogenesis when cocultured with fibroblasts from ER-alpha(-)/PgR(-) or ER-alpha(+)/PgR(+) breast tumors; however, EIII8 cells cocultured with ER-alpha(-)/PgR(-) tumor-derived fibroblasts exhibited decreased tamoxifen sensitivity compared with cells cocultured with ER-alpha(+)/PgR(+) tumor fibroblasts. Fibroblast-induced tamoxifen resistance was accompanied by mitogen-activated protein kinase and Akt hyperactivation, reduced sensitivity to U0126 or LY294002, and ER-alpha hyperphosphorylation in the activation function-1 domain. The intrinsic tamoxifen resistance of EIII8-Tam(R) cells correlated with constitutive ER-alpha hyperphosphorylation that was unaffected by the tumor fibroblasts. Our results suggest that tumor fibroblast-induced tamoxifen resistance of EIII8 cells is not mediated by epidermal growth factor receptor or insulin-like growth factor (IGF)-1R axes because no correlation was found between expression levels of IGF-1, IGF-2, phosphorylated IGF-1R, or epidermal growth factor receptor, and tamoxifen sensitivity of EIII8 fibroblast cultures.